Interactions in the niche between hematopoietic stem cells (HSC), osteoblasts (OB), mesenchymal stem cells (MSC), and endothelial cells (EC) are critical for HSC self-renewal and maintenance of the stem cell pool. However, molecules mediating these interactions are not well defined. Our published data determined that CD166, a member of the immunoglobulin superfamily, is expressed on normal murine HSC and niche OB. More recently, we determined that CD166 is also expressed on putative MSC and bone marrow (BM)-derived EC. With its homophilic cell-cell interactions, CD166 may be both ligand and receptor of HSC in their interactions with all these elements of the hematopoietic niche. We published that CD166 identifies a very small group of murine BM cells (LSKCD48-CD166+CD150+) highly enriched for HSC function. More than 90% of long-term in vivo repopulating potential of human cord blood-derived cells is restricted to CD34+CD38- Lineage- cells that coexpress CD166. Engraftment of HSC from CD166-/- mice in lethally irradiated hosts is defective and the CD166-/- niche cannot support the long-term engraftment of repopulating HSC. CD166 expression on STAT3-/- LSKCD48-CD150+ cells is significantly reduced and inhibition of STAT3 concomitantly reduces CD166 expression. Engraftment of STAT3-/- HSC is significantly suppressed in 1o and 2o recipients and STAT3 binds to the CD166 promoter suggesting that both CD166 and STAT3 are functionally coupled and involved in HSC competence. We hypothesize that CD166 is a universal functional marker required for HSC-niche interactions that support engraftment and HSC self-renewal and that CD166-mediated control of HSC self-renewal can be manipulated via signaling pathways impacted by STAT3 to enhance HSC clinical efficacy. Our two specific aims will: 1) Test the hypothesis that CD166 expression on HSC and on OB, MSC, and EC of the hematopoietic niche is required for the maintenance of stem cell function and that loss of CD166 expression on HSC or any of these cellular elements of the hematopoietic niche is detrimental to HSC function and 2) Test the hypothesis that gp130-activated STAT3 regulates CD166 expression and the engraftment of HSC, and that therapeutic targeting of CD166 expression can be translated to enhance human HSC engraftment in the setting of cord blood transplantation. The significance of this proposal is that it will examine the role of a new and unique functional marker of human and murine HSC and will investigate the impact of CD166 modulation on stem cell function. The conserved murine and human CD166 homology facilitates efficient translational investigations aimed at enhancing HSC engraftment and clinical utility. The novelty of this proposal stems from its potential to establish the functional capacity of a marker present on both murine and human HSC and on at least 3 cell types of the HN. The proposed research is innovative because it implicates STAT3 in the regulation of CD166 expression and will explore the molecular mechanism through which a new pathway involving CD166 and STAT3 controls HSC function, self-renewal, and engraftment.